open access

Vol 45, No 4 (2007)
ORIGINAL PAPERS
Published online: 2008-01-01
Submitted: 2011-12-19
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Ex vivo measurement of calpain activation in human peripheral blood lymphocytes by detection of immunoreactive products of calpastatin degradation.

Anna Mikosik, Anna Zaremba, Zofia Puchalska, Agnieszka Daca, Zaneta Smolenska, Paulina Lopatniuk, Andrzej Mital, Andrzej Hellman, Ewa Bryl, Jacek M Witkowski
Folia Histochem Cytobiol 2007;45(4):343-347.

open access

Vol 45, No 4 (2007)
ORIGINAL PAPERS
Published online: 2008-01-01
Submitted: 2011-12-19

Abstract

Limited proteolysis of multiple intracellular proteins by endogenous Ca-dependent cysteine proteases--calpains--is an important regulatory mechanism for cell proliferation, apoptosis etc. Its importance for cellular functions is stressed by existence of endogenous calpain inhibitors--calpastatins. The calpain-calpastatin system within living cells is in a fragile balance, which depends on both partners. The interdependence of calpain--a protease--and calpastatin--an endogenous inhibitor and at the same time a substrate for this enzyme makes any assessment of actual activity of this enzyme in the cells very difficult. In this work we made an attempt to estimate and compare the activity of calpain in human peripheral blood lymphocytes by assessing the levels of limited proteolysis of calpastatin in these cells by western blot, while at the same time the levels of calpain protein inside these cells was measured by flow cytometry. Our results indicate that it is possible to compare (semi-quantitatively) the activities of calpain in peripheral blood CD4+ and CD19+ lymphocytes from various donors that way. Preliminary results showed that calpain activity is increased in the CD4+ T cells isolated from peripheral blood of rheumatoid arthritis patients as compared to control lymphocytes. Extremely high intrinsic activity of calpain was detected in chronic lymphocytic leukemia (CD19+) cells. All this confirms the detection of immunoreactive products of calpastatin as a good maker of endogenous calpain activity.

Abstract

Limited proteolysis of multiple intracellular proteins by endogenous Ca-dependent cysteine proteases--calpains--is an important regulatory mechanism for cell proliferation, apoptosis etc. Its importance for cellular functions is stressed by existence of endogenous calpain inhibitors--calpastatins. The calpain-calpastatin system within living cells is in a fragile balance, which depends on both partners. The interdependence of calpain--a protease--and calpastatin--an endogenous inhibitor and at the same time a substrate for this enzyme makes any assessment of actual activity of this enzyme in the cells very difficult. In this work we made an attempt to estimate and compare the activity of calpain in human peripheral blood lymphocytes by assessing the levels of limited proteolysis of calpastatin in these cells by western blot, while at the same time the levels of calpain protein inside these cells was measured by flow cytometry. Our results indicate that it is possible to compare (semi-quantitatively) the activities of calpain in peripheral blood CD4+ and CD19+ lymphocytes from various donors that way. Preliminary results showed that calpain activity is increased in the CD4+ T cells isolated from peripheral blood of rheumatoid arthritis patients as compared to control lymphocytes. Extremely high intrinsic activity of calpain was detected in chronic lymphocytic leukemia (CD19+) cells. All this confirms the detection of immunoreactive products of calpastatin as a good maker of endogenous calpain activity.
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About this article
Title

Ex vivo measurement of calpain activation in human peripheral blood lymphocytes by detection of immunoreactive products of calpastatin degradation.

Journal

Folia Histochemica et Cytobiologica

Issue

Vol 45, No 4 (2007)

Pages

343-347

Published online

2008-01-01

Bibliographic record

Folia Histochem Cytobiol 2007;45(4):343-347.

Authors

Anna Mikosik
Anna Zaremba
Zofia Puchalska
Agnieszka Daca
Zaneta Smolenska
Paulina Lopatniuk
Andrzej Mital
Andrzej Hellman
Ewa Bryl
Jacek M Witkowski

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